Two promising cases for obtaining a deeper understanding of the relation of active center structure to function will be the main (and continuing) objects of study. One involves a single-chain enzyme, pancreatic ribonuclease, and utilizes a comparative approach: members of the known homologous series of this protein in the pancreas of vertebrates are, or will be, purified. Amino acid sequence determination, positions and neighbors of active center residues, quantitative catalytic differences and physical properties, will be compared in suitable cases. This study will be aimed to give insights into (a) the absolute requirements for the active center structure; (b) the effects thereon of natural mutations; (c) the extent and types of change tolerated by a single-chain hydrolase structure in the course of evolution. The second case involves a multi-subunit enzyme, yeast hexokinase. The known subunit structure (2 identical subunits) will be probed with respect to the chemical features of binding and catalytic sites thereon, and the interactions between the subunits. Specific active-center directed reactivities will be used, as well as determination of the catalytic properties of the subunits alone and in the oligomeric assembly. Relation of the activity to subunit association, and related physicochemical properties will also be employed as an approach. Isoenzyme differences will be considered. The amino acid sequence determination will be pursued. The main aim will be to relate the sequence and active center properties to the three-dimensional structure now being elucidated by X-ray crystallography by a co-operative effort elsewhere.